The objective here was to define the small intestinal mucosal genes whose expressions were affected in coeliac disease. This was done by comparing the gene expression between untreated coeliac disease patients and healthy controls, between untreated and treated coeliac disease patients and finally between treated coeliac disease patients and healthy controls (II).
Those genes whose transcription was altered in the same direction and whose mean ratio was above 1.25, were considered significant. Thus it was hoped to gain a result that was true for the whole patient group, not only for one patient.
There were 156 genes which were changed in untreated coeliac disease compared to healthy controls. When untreated coeliac disease subjects were compared to treated, 98 genes were found to be affected. When treated coeliac patients were compared to healthy controls, 60 were found to be affected.
Altogether 263 of the arrayed 5184 genes or ESTs were affected.
All significantly altered genes were grouped into ontology classes. Of all significantly altered genes 20 (8 %) were involved in metabolism other than energy metabolism (Supplementary data, Appendix 2, group A). All the genes belonged to different metabolic pathways. Only five (2 %) genes belonged to the subgroup of metabolism, the generation of precursor metabolites and energy
consisted 33 (13 %) of affected genes (Supplementary data, Appendix 2, group D).
In all coeliac groups DLX4, a homeobox gene, was the only gene to be up-regulated. These were 39 (14 %) genes having effect on the cell signal transducer activity (Supplementary data, Appendix 2, group E). The EGFR gene and two clones of the related to the N terminus of tre (RENTRE) were up-regulated in untreated coeliac disease (untreated coeliac disease compared to healthy control, and untreated coeliac disease compared to treated coeliac disease). The B cell marker, MS4A1, was up-regulated in coeliac disease patients (untreated coeliac disease vs. healthy control and treated coeliac disease vs. healthy control). The expression of β-catenin was up-regulated in untreated coeliac disease when compared to healthy controls, but that of E-cadherin remained unchanged. The genes that participated in protein folding and modification, i.e. cellular macromolecule metabolism comprised 11 (4 %) of all affected genes (Supplementary data, Appendix 2, group F). Betaine-homocysteine methyltransferase 2 (BHMT2), which is activated by a low folate concentration was up-regulated only in treated coeliac disease compared to healthy controls.
As coeliac disease is an HLA-linked inflammatory disorder, the cell rescue and defence associated genes were scrutinised (Supplementary data, Appendix 2, group G). The HLA-DQB1 gene was up-regulated in untreated coeliac disease when compared to the other study groups. Other potentially interesting genes were interleukin receptor-18 gene (IL-18R1), belonging to the interleukin 1 receptor family, and protein tyrosine phosphatase, receptor type, C-associated protein gene (PTPRCAP), which is a key regulator of T- and B-lymphocyte activation. Both of these were down-regulated in untreated coeliac disease as compared to healthy controls; 21 out of 263 (8 %) of genes belonged to group that controls cellular organisation (Supplementary data, Appendix 2, group H).
The expressions of matrix metalloproteinase (MMP) 1 and 2 mRNAs and metalloproteinase inhibitor mRNA (TIMP-1) were so low that the intensity was at the same level as the background labelling. The genes affecting processes related to transport and transport facilitation, comprising 14 (5 %) of all significantly altered genes, were up-regulated in coeliac disease patients (Supplementary data, Appendix 2, group I). Especially the transcription of several ion pumps such as ATPase, H+ transporting (ATP6V0A1), ATPase, Na+/K+ transporting (ATP1B3) and ATPase, Ca++ transporting (ATP2A2) were up-regulated. Altogether 96 out of 263 of the altered genes or ESTs (36.5%) have no function known hitherto (Supplementary data, Appendix 2, group J).
The genes located in the coeliac disease predisposition regions (5q31-33, 2q31-33 and 15q11-13) were also scrutinised (Table 6. in II), but none present on the used filter exhibited significant change in gene expression.
The genes evincing significantly altered expression in coeliac disease, which
aetiology of coeliac disease. There were only eight such genes: they are listed in Table 7.
Validation of data was made with qRT-PCR for three selected genes chosen from different functional classes. The expression of two inflammatory genes IFNγ and IL-10, which are known to undergo a significant up-regulation in their expression in coeliac disease small intestinal biopsies (Forsberg et al. 2002), were assessed (Table 8). The expression changes detected with cDNA microarray (Supplementary data, Appendix 2) were all in concordance with qRT-PCR.
Table7.List of genesevincing significantly altered transcription independent of a gluten-free diet. nameGenBank nochr location UntreatedCD vs. Healthy control Untreated CD vs. Treated CD
Treated CD vs. Healthy control DLX4: distal-less homeobox 4R9249517q21.331.951.421.37 ADAMTS3: a disintegrin-like and metalloproteaseAA4027604q13.30.590.61 PTDSS1: phosphatidylserine synthase 1H289848q220.750.81 MS4A1: membrane-spanning 4-domains, subfamily A, member 1
N9138511q121.741.46 Homo sapiens cDNA FLJ11723 fis, clone HEMBA1005314
T8408411p140.600.63 DPAGT1: dolichyl-phosphate (UDP-N- acetylglucosamine) N- acetylglucosaminephosphotransferase 1 (GlcNAc-1-P transferase) R5561911q23.30.440.70 ARNT2: aryl-hydrocarbon receptor nuclear translocator 2
AA01977415q240.410.53 FXR2: fragile X mental retardation, autosomal homolog 2
AA48972917p13.10.600.70 een untreated and treated coeliac disease (CD) patients and healthy controls. Upward arrowdenotes up-regulated NA expression, downward arrow down-regulated mRNA expression.Ratios are mean values calculated from three separate microarray ents.
8.mRNA levels of IFNγ, IL-10, MS4A1, DIO2, KbRas in small intestinal biopsiesdetected with cDNA microarray and qRT-PCR. nameGenBank no Microarray UntreatedCD vs Healthy control qRT-PCR UntreatedCD vs. Healthy control Microarray UntreatedCD vs. TreatedCD qRT-PCR UntreatedCD vs. TreatedCD Microarray TreatedCD vs.Healthy control
qRT-PCR TreatedCD vs.Healthy control IFNγn.e.519.83n.e.96.41n.e.5.39 IL-10n.e.1.68n.e.1.23n.e.1.37 MS4A1N913851.748.951.151.951.464.59 DIO2R622420.710.550.690.131.274.31 KbRASR631720.91.080.750.161.526.70 MMP1AA143201u.d.u.d.u.d.u.d.u.d.u.d. pward arrowdenotes up-regulated mRNA expression, downward arrow down-regulated mRNA expression, bold indicates genes significant alteration in expression. n.e. = not existent, not arrayed on filter; u.d. = undetectable transcript.Ratios are mean values calculated from experiments and five separateqRT-PCR experiments.